In vitro nucleic acid amplification techniques have provided powerful tools for detection and analysis of small amounts of nucleic acids. The extreme sensitivity of such methods has lead to attempts to develop them for diagnosis of infectious and genetic diseases, isolation of genes for analysis, and detection of specific nucleic acids as in forensic medicine. Nucleic acid amplification techniques can be grouped according to the temperature requirements of the procedure. The polymerase chain reaction (PCR; R. K. Saiki, et al. 1985. Science 230, 1350-1354), ligase chain reaction (LCR; D. Y. Wu, et al. 1989. Genomics 4, 560-569; K. Barringer, et al. 1990. Gene 89, 117-122; F. Barany. 1991. Proc. Natl. Acad. Sci. USA 88, 189-193) and transcription-based amplification (D. Y. Kwoh, et al. 1989. Proc. Natl. Acad. Sci. USA 86, 1173-1177) require temperature cycling. In contrast, methods such as strand displacement amplification (SDA; G. T. Walker, et al. 1992. Proc. Natl. Acad. Sci. USA 89, 392-396; G. T. Walker, et al. 1992. Nuc. Acids. Res. 20, 1691-1696), selfsustained sequence replication (3SR; J. C. Guatelli, et al. 1990. Proc. Natl. Acad. Sci. USA 87, 1874-1878) and the Q.beta. replicase system (P. M. Lizardi, et al. 1988. BioTechnology 6, 1197-1202) are isothermal reactions. In addition, WO 90/10064 and WO 91/03573 describe use of the bactefiophage phi29 replication origin for isothermal replication of nucleic acids.
In general, diagnosis and screening for specific nucleic acids using nucleic acid amplification techniques has been limited by the necessity of amplifying a single target sequence at a time. In instances where any of multiple possible nucleic acid sequences may be present (e.g., infectious disease diagnosis), performing multiple separate assays by this procedure is cumbersome and time-consuming. U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,800,159 describe the PCR. Although these inventors state that multiple sequences may be detected, no procedure for amplifying multiple target sequences simultaneously is disclosed. When multiple target sequences are amplified, it is by sequentially amplifying single targets in separate PCRs. In fact, when multiple pairs of primers directed to different target sequences are added to a single PCR, the reaction produces unacceptably high levels of nonspecific amplification and background. An improvement on the PCR which reportedly allows simultaneous amplification of multiple target sequences is described in published European Patent Application No. 0 364 255. This is referred to as multiplex DNA amplification. In this method, multiple pairs of primers are added to the nucleic acid containing the target sequences. Each primer pair hybridizes to a different selected target sequence, which is subsequently amplified in a temperature-cycling reaction similar to PCR.
Certain nucleic acid amplification procedures have employed addition of defined sequences to the ends of nueleic acid fragments prior to amplification. U.S. Pat. No. 5,104,792 describes a modification of PCR which allows amplification of nucleic acid fragments for which the sequence is not known. The primers for the amplification reaction contain random degenerate sequences at their 3' ends and a defined sequence at their 5' ends. Extension of the primers produces fragments containing unknown sequences which are flanked by the defined sequence. These fragments may then be amplified in a conventional PCR using primers which hybridize to the known flanking sequence. Another method for PCR amplification of unknown DNA which flanks a known sequence is described by D. H. Jones and S. C. Winistorfer (1992. Nuc. Acids. Res. 20, 595-600, "panhandle PCR"). In panhandle PCR, a single-stranded oligonucleotide complementary to a sequence in the known DNA is ligated to the 3' ends of a double stranded fragment. Upon denaturation and intrastrand reannealing, the complementary sequences hybridize and the recessed 3' end is extended with polymerase, producing the unknown sequence flanked by the known sequence. The known sequence can then be used to prepare primers for amplification of the unknown sequence. Similar methods for generation of a hairpin structure and single primer amplification are described in published European Patent Application No. 0 379 369. WO 90/09457 describes a sequence-independent method for amplification of DNA sequences which are entirely unknown. Universal oligonucleotide primer pairs are ligated to the target DNA by blunt-end ligation so that PCR. amplification may be primed using these known primers.
Several methods are known which allow amplification of target sequences when only partial sequence information is known. A. R. Shuldiner, et al. (1990. Gene 91, 139-142) describe a modification of reverse transcription PCR in which a unique sequence is appended to the 5' end of the first strand during reverse transcription. First strand synthesis is primed by a hybrid primer which is complementary to the RNA target at the 3' end and contains the unique sequence at the 5' end. The cDNA is then amplified using a primer directed to the unique sequence and a primer directed to a target-specific sequence. This reportedly reduces amplification of carryover contaminants. Published European Patent Application No. 0 469 755 discloses a method for producing single stranded polynucleotides having two segments that are non-contiguous and complementary. A sequence complementary to an existing sequence in the polynucleotide is introduced by extension of a primer which hybridizes to the polynucleotide at its 3' end and has the complement of the existing sequence at its 5' end. After extension of the primer the polynucleotide can be amplified using a single primer. V. Shyamala and G. F. L. Ames (1989. Gene 84, 1-8) teach a method for PCR amplification of DNA when the sequence of only one end is available (SSP-PCR). The unknown end is ligated to a genetic vector sequence, and the fragment is amplified using a gene-specific primer and a generic vector primer. Similar methods are disclosed in Published European Patent Application No. 0 356 021. WO 90/01064 describes amplification of a sequence by synthesizing a complementary strand primed with a sequence-specific primer directed to a known portion of the sequence. A homopolymer is added to the 3' end of the complement and the sequence is amplified using a homopolymer primer and a primer which is homologous to a region of the sequence-specific primer. Adaptation of PCR to footprinting is taught by P. R. Mueller and B. Wold (1989. Science 246, 780-786). For footprinting, a common oligonucleotide sequence is ligated to the unique end of each fragment of the footprint ladder. The fragments are amplified using a primer complementary to the common sequence and a primer complementary to the known sequence of the fixed end.
The present methods provide a means for appending any adapter sequence or any pair of adapter sequences to any target prior to amplification by primer extension. The adapter sequences reduce the number of specific primers which are required for simultaneous amplification of two or more target sequences in a single primer extension amplification reaction (referred to herein as "multiplex amplification" or "multiplexing"). Conventional multiplexing involves putting into the reaction primers specific for amplification of each target sequence, i.e., each target is amplified by a specific primer or pair of primers. Conventional multiplexing provides satisfactory results in certain circumstances, but has drawbacks in that multiple specific primers must be prepared for each multiplex reaction. Often, however, multiple sequences cannot be readily amplified and detected using conventional multiplexing due to generation of high levels of nonspecific background amplification. The adapter mediated multiplexing of the invention is an alternative to conventional multiplexing which gives improved results in certain cases. Of the foregoing publications, only EPO 0 364 255 and Mueller and Wold address the problem of simultaneously amplifying multiple target sequences. Both teach simultaneous amplification for PCR, which in part due to its temperature cycling provides significantly different reaction conditions as compared to isothermal amplifications such as SDA. Although certain of the foregoing publications describe appending defined sequences to either end of a fragment prior to amplification, the addition of the defined end and amplification are performed in separate reactions. Further, the present invention for the first time provides methods for simultaneously amplifying multiple target sequences by SDA without the necessity of providing separate specific primers for each target. The inventive methods are particularly advantageous in that addition of defined adapter sequences to the ends of the target sequences and the amplification reaction occur in a single reaction mix and appear as a single step to the practitioner.